These are the general mammalian cell passaging rules, they work for CHO cells and HEK cells.

The cells should be left to grow for two days, after what their density should be reduced and the medium should be changed.

We usually bring them either to 2 mio/ml, either to 1 mio/ml.

• Put the volume of medium that corresponds to the amount of cells you'd like to passage at 37°C, leave it there to heat for 10 minutes (more for bigger volumes, less for smaller).
• Measure the PCV of your current cell sample. Usually, the cells double every day, so you can estimate their number, but a PCV is more precise. Take 200 µl of cell suspension in a mini-PCV tube, centrifuge them (1 min at 5000 rpm), the cells will fall down in the thin tube at the bottom. Use the VoluPAC Reader to measure the PCV (the volume in µl the centrifuged cells take).
  • If you're dealing with HEK cells, the PCV is also the number of millions of cells/ml you have.
  • If you have CHO cells, divide the PCV by 0.4 to obtain the mio/ml cell density.
• Take the volume that will contain the amount of cells you need from the original sample in a centrifuge tube, centrifuge it at 1500 rpm for 3 minutes. A pellet will form.
• Remove the supernatant (under the hood) either with the vacuum pump, either by simply discarding it.
• Resuspend in 10 ml of warmed medium (pipet up and down)
• Add the remaining medium, mix again
• Split the cultures into 10 ml samples in separate tubes, put them in the shaker at 37°C, passage again after 2 days.